Werner syndrome (WS) is a hereditary premature aging disorder characterized by chromosomal instability. The WRN gene product is a helicase/exonuclease that presumably functions in DNA metabolism to preserve genome integrity. To understand the DNA structures and cellular pathways that WRN impacts, we have systematically examined the DNA substrate preferences of WRN helicase for unwinding and its interactions with human nuclear proteins. Our biochemical studies indicate that WRN preferentially unwinds DNA replication structures in a defined orientation and utilizes specific DNA structures for recognition. We have initiated a kinetic analysis of WRN helicase activity to define the mechanism of DNA unwinding. To further understand its molecular functions, we have characterized the functional interaction of WRN protein with human Flap Endonuclease 1 (FEN-1), a structure-specific nuclease implicated in DNA repair, replication, and recombination. The results indicate that WRN stimulates FEN-1 cleavage of important DNA intermediates by a unique mechanism whereby the efficiency of FEN-1 cleavage is dramatically enhanced. Our results indicate that WRN activates FEN-1 resolution of a key DNA structure of homologous recombination, the four-stranded Holliday Junction. These findings suggest a direct role of WRN protein to facilitate the resolution of recombination intermediates by functionally interacting with human resolvases. The aberrant mitotic recombination and genomic instability in WS cells arises from inappropriate processing of recombination intermediates that are a consequence of a defect in the restablishment of the replication fork after arrest.